CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Nas infecÃÃes experimentais, estudos utilizando inÃculo purificado de Leishmania, com populaÃÃes metacÃclicas mais homogÃneas, tem sido importantes por se aproximarem mais da condiÃÃo de infecÃÃo natural e por eliminar uma possÃvel resposta inflamatÃria induzida por restos parasitÃrios. A superfÃcie dos vÃrios parasitos reveste-se por diversos glicoconjugados. Estes, por sua vez, podem ser reconhecidos especifica e reversivelmente por lectinas, proteÃnas que possuem afinidade a carboidratos. Por conta disso, tem-se tentado purificar promastigotas metacÃclicas de culturas de Leishmania atravÃs de aglutinaÃÃo mediada por lectinas. Com esse intuito, buscou-se avaliar se lectinas de diferentes especificidades seriam capazes de aglutinar promastigotas de L. amazonensis em diferentes estÃgios evolutivos. Para isso, foi utilizado um painel de 7 lectinas, e a especificidade de ligaÃÃo aos carboidratos de superfÃcie foi analisada atravÃs de ensaios de aglutinaÃÃo, sendo a lectina de Dioclea violacea (DVL) selecionada para a realizaÃÃo dos ensaios in vivo. A DVL foi capaz de aglutinar L. amazonensis num percentual de 78% em cultura de fase logarÃtmica e 52% em fase estacionÃria. Em seguida, promastigotas de fase estacionÃria foram incubadas com DVL para avaliar se a aglutinaÃÃo era estÃgio-especifica. Posteriormente, realizou-se infecÃÃo experimental em hamsters dourados (106 promastigotas/animal) para avaliar a infectividade das fraÃÃes purificadas. Os animais foram divididos em 3 grupos: FraÃÃo aglutinada (FA) (n=8); FraÃÃo nÃo aglutinada (FNA) (n=9); Controle positivo (CT) (n=8). A evoluÃÃo da infecÃÃo foi acompanhada pelo tamanho da lesÃo, nas 6 semanas pÃs-infecÃÃo e pela quantificaÃÃo da carga parasitÃria no linfonodo regional, por diluiÃÃo limitante, bem como pela anÃlise anatomopatolÃgica das lesÃes. Em todos os grupos as lesÃes iniciaram na 3 semana. O tamanho da lesÃo foi muito semelhante em todos os grupos, com exceÃÃo da 4 e 5 semanas, onde FNA apresentou uma variaÃÃo transitÃria (p<0,01). Quanto à carga parasitÃria e Ãs alteraÃÃes histopatolÃgicas nÃo houve diferenÃa entre os grupos avaliados. Esses dados sugerem que, embora a DVL seja capaz de se ligar especificamente aos glicoconjugados de superfÃcie das promastigotas, induzindo importante poder de aglutinaÃÃo nas duas fases de crescimento, ela nÃo foi capaz de selecionar formas infectantes de L. amazonensis. / In experimental leishmaniasis infections, the use of metacyclic enriched inoculum is very important because it can simulate the natural infection and avoids the inflamatory response induced by the high density parasite inoculum. The surface of different evolutionary forms of Leishmania is coated by several glycoconjugates that can be recognized specifically and reversibly by lectins, proteins that have affinity with carbohydrates. This being the case, one could use lectins in order to purify metacyclic Leishmania. The aim of the current study was to evaluate whether lectins of different specificities would agglutinate L. amazonensis promastigotes of different evolutionary forms. A panel of 7 lectins were used. The binding specificity was analyzed by agglutination tests. DVL agglutinated 78% of L. amazonensis promastigotes from logarithmic phase culture and 52% from stationary phase, and therefore was selected for in vivo tests. Stationary phase promastigotes were incubated with DVL to evaluate if the agglutination was stage-specific and it was purified the agglutinated and non agglutinated fractions. Golden hamsters were infected with 106 promastigotes and grouped as: Agglutinated fraction (FA) (n=8); Non agglutinated fraction (FNA) (n=9); Control (CT) (n=8). The lesion size was measured over the course of 6 weeks. The parasite load of regional lymph node was quantified by limiting dilution and histopathological analysis of the lesions were performed on their paws. The lesions began at the third week in all groups. The lesionâs size was similar in all of them, except at the fourth and fifth weeks that FNA presented a transitory reduction (p<0,01). There were no significant differences concerning the parasite load and histopathologic changes among the groups. These data suggest that DVL did not select effectivelly infective forms of L. amazonensis, although it agglutinates promastigotes from the two culture growth phases.

No description available.

This thesis consists of two parts. The first part is concerned with further structural and related studies of jacalin, one of the two lectins found in jack fruit seeds. The second part deals with the search of mycobacterial and archeal genomes for lectins. The β-prism I fold was identified as a lectin fold through the X-ray analysis of jacalin way back in 1996. Subsequent structural studies on jacalin are described in the first chapter in context of the overall efforts on lectins with particular reference to those on lectins with β-prism I fold. The structure of jacalin has been thoroughly characterized through the analysis of several crystals. The extended binding site of the lectin, made up of the primary binding site and secondary sites A and B, has also been characterized through studies on different jacalin-sugar complexes. However, nuances of jacalin-carbohydrate interactions remain underexplored with respect to two specific issues. The first issue is concerned with the structural basis for the lower affinity of jacalin for β-substituted sugars. The second has to do with the influence of the anomeric nature of the glycosidic linkage on the location of the reducing and non-reducing sugars in disaccharides when interacting with jacalin. Part of the work described in the thesis addresses these two issues. It was surmised that the lower affinity of β-galactosides to jacalin as compared to α-galactosides, is caused by steric interactions of the substituents in the former with the protein. This issue is explored both energetically and structurally in Chapter 2 using appropriately derivatized monosaccharide complexes of jacalin. It turns out that the earlier surmise is not correct. The interactions of the substituent with the binding site remain essentially the same irrespective of the anomeric nature of the substitution. This is achieved through a distortion of the sugar ring in β-galactosides. The difference in energy, and therefore affinity, is caused by the distortion of the sugar ring in β-galactosides. The elucidation of this unprecedented distortion of the ligand as a strategy for modulating affinity is of general interest. The crystal structures also provide a rationale for the relative affinities of the different carbohydrate ligands to jacalin. The crystal structures of jacalin complexed with α-linked oligosaccharides Gal α-(1,4) Gal and Gal α-(1,3) Gal β-(1,4) Gal, as described in Chapter 3, have been determined with the primary objective of exploring the effect of linkage on the location of reducing and non-reducing sugars in the extended binding site of the lectin, an issue which has not been studied thoroughly. Contrary to the earlier surmise based on simple steric considerations, the two structures demonstrate that α-linked sugars can bind to jacalin with non-reducing sugar at the primary binding site. This is made possible substantially on account of the hitherto underestimated plasticity of a non-polar region of the extended binding site. Modelling studies involving conformational search and energy minimization, along with available crystallographic and thermodynamic data, indicate a strong preference for complexation with Gal β-(1,3) Gal with the reducing Gal at the primary site, followed by that with Gal α-(1,3) Gal, with the reducing or non-reducing Gal located at the primary binding site. This observation is in consonance with the facility of jacalin to bind mucin type O-glycans containing T-antigen core. Crystal structures of jacalin in complex with GlcNAc β-(1,3) Gal-β-OMe and Gal β-(1,3) Gal-β-OMe have also been described in Chapter 4. The binding of the ligands to jacalin is similar to that of analogous α-substituted disaccharides. However, the β-substituted β-(1,3) linked disaccharides get distorted at the anomeric centre and the glycosidic linkage. The distortion results in higher internal energies of the ligands leading to lower affinity to the lectin. This confirms the possibility of using ligand distortion as a strategy for modulating binding affinity. Unlike in the case of β-substituted monosaccharides bound to jacalin, where a larger distortion at the anomeric centre was observed, smaller distortions are distributed among two centres in the structures of the two β-substituted β-(1,3) linked disaccharides presented here. These disaccharides, like the unsubstituted and α-substituted counterparts, bind jacalin with the reducing Gal at the primary binding site, indicating that the lower binding affinity of β-substituted disaccharides is not enough to overcome the intrinsic propensity of Gal β-(1,3) Gal based disaccharides to bind jacalin with the reducing sugar at the primary site. Although originally isolated from plants, lectins were also found subsequently in all forms of life, including bacteria. Studies on microbial lectins have not been as extensive as on those from plants and animals, although there have been some outstanding individual investigations on bacterial toxins like ADP-ribosylating toxins and neurotoxins. In addition to bacterial toxins, adhesins, β-trefoil lectins and cyanobacterial lectins form other important subgroups which have been explored using crystallography. Features pertaining to their three dimensional folds, carbohydrate specificity and biological properties are described in Chapter 5, to set the stage for the work discussed in the second part of the thesis. Studies on mycobacterial lectins were unexplored until work was initiated in the area in this laboratory some years ago. One of the lectins, identified on the basis of a bioinformatics search of M. tuberculosis H37Rv genome was cloned, expressed and crystallized. Also cloned, expressed and crystallized is another lectin from M. smegmatis. Biophysical and modelling studies were carried out on the full length protein containing this lectin. However, systematic efforts on mycobacterial lectins were conspicuous by their absence. The first chapter (Chapter 6) in the second part of the thesis is concerned with a genomic search for lectins in mycobacterial genomes. It was also realized that hardly anything is known about archeal lectins. Therefore, as discussed in the final chapter, a genomic search for archeal lectins was undertaken. Sixty-four sequences containing lectin domains with homologs of known three-dimensional structure were identified through a search of mycobacterial genomes and are described in detail in Chapter 6. They appear to belong to the β-prism II, the C-type, the Microcystis virdis (MV), and the β-trefoil lectin folds. The first three always occur in conjunction with the LysM, the PI-PLC, and the β-grasp domains, respectively while mycobacterial β-trefoil lectins are unaccompanied by any other domain. Thirty heparin binding hemagglutinins (HBHA), already annotated, have also been included in the study although they have no homologs of known three-dimensional structure. The biological role of HBHA has been well characterized. A comparison between the sequences of the lectin from pathogenic and non-pathogenic mycobacteria provides insights into the carbohydrate binding region of the molecule, but the structure of the molecule is yet to be determined. A reasonable picture of the structural features of other mycobacterial proteins containing one of the four lectin domains can be gleaned through the examination of homologous proteins, although the structure of none of them is available. Their biological role is yet to be elucidated. The work presented here is among the first steps towards exploring the almost unexplored area of the structural biology of mycobacterial lectins. As mentioned in Chapter 7, forty six lectin domains, which have homologues among well established eukaryotic and bacterial lectins of known three dimensional structure, have been identified through a search of 165 archeal genomes using a multi-pronged approach involving domain recognition, sequence search and analysis of binding sites. Twenty one of them have the 7-bladed β-propeller lectin fold while 16 have the β-trefoil fold and 7 the legume lectin fold. The remainder assumes the C-type lectin, the β-prism I and the tachylectin folds. Acceptable models for almost all of them could be generated using the appropriate lectins of known three dimensional structure as templates, with binding sites at one or more expected locations. The work represents the first comprehensive bioinformatics study of archeal lectins. The presence of lectins with the same fold in all domains of life indicates their ancient origin well before the divergence of the three branches. Further work is necessary to identify archeal lectins which have no homologues among eukaryotic and bacterial species.

Yu Yun Lung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 101-114). / Abstracts in English and Chinese. / Acknowledgments / Abstract / Table of Contents / Chapter CHAPTER 1 --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- General Structure of Lectins --- p.1 / Chapter 1.1.1 --- Metal Binding Sites --- p.2 / Chapter 1.1.2. --- Hydrophobic Sites --- p.3 / Chapter 1.1.3. --- Glycosylation Sites --- p.3 / Chapter 1.2 --- Carbohydrate Specificities of Lectins --- p.4 / Chapter 1.3 --- Plant Lectins --- p.4 / Chapter 1.3.1 --- Localization of lectins in plants --- p.4 / Chapter 1.3.1.1 --- Localization in seeds --- p.4 / Chapter 1.3.1.2 --- Localization in vegetative parts --- p.5 / Chapter 1.3.1.3 --- Biosynthesis of plant lectins --- p.6 / Chapter 1.3.2 --- Functions of plant lectins in plants --- p.7 / Chapter 1.3.2.1 --- In cell growth --- p.7 / Chapter 1.3.2.2 --- In storage --- p.8 / Chapter 1.3.2.3 --- In plant defence --- p.8 / Chapter 1.3.2.4 --- In nitrogen cycle --- p.10 / Chapter 1.3.3 --- Biological activities of plant lectins in other organisms --- p.13 / Chapter 1.3.3.1 --- Immunomodulatory activity --- p.13 / Chapter 1.3.3.2 --- Antitumor and antiproliferative activities --- p.14 / Chapter 1.3.3.3 --- Mitogenic activity --- p.14 / Chapter 1.3.3.4 --- Antiviral activity --- p.14 / Chapter 1.3.4 --- Relationship between lectins and ribosome inactivating proteins: family of ricin-related proteins --- p.16 / Chapter 1.3.5 --- Applications of plant lectins --- p.18 / Chapter 1.3.5.1 --- In scientific research --- p.18 / Chapter 1.3.5.2 --- In medical research --- p.19 / Chapter 1.4 --- Animal Lectins --- p.20 / Chapter 1.4.1 --- Some properties of animal lectins --- p.20 / Chapter 1.4.2 --- Functions of animal lectins --- p.22 / Chapter 1.4.2.1 --- In protein metabolism --- p.22 / Chapter 1.4.2.2 --- As a mediator of binding and phagocytosis of microorganisms --- p.22 / Chapter 1.4.2.3 --- Control of differentiation and organ formation --- p.23 / Chapter 1.4.2.4 --- Lectins and migration of lymphocytes --- p.23 / Chapter 1.4.2.5 --- Lectins and metastasis --- p.24 / Chapter 1.5 --- Mushroom lectins --- p.25 / Chapter 1.6 --- Regulation of lectins --- p.29 / Chapter 1.7 --- Isolation and purification of lectins --- p.31 / Chapter 1.8 --- Objectives of the present study --- p.33 / Chapter CHAPTER 2 --- "SCREENING FOR HEMAGGLUTINATING ACTIVITY IN EXTRACTS OF SEEDS, FRUITS, VEGETABLES AND CHINESE MEDICINAL HERBS" --- p.35 / Chapter 2.1 --- Introduction --- p.35 / Chapter 2.2 --- Materials and methods --- p.36 / Chapter 2.3 --- Results --- p.38 / Chapter 2.4 --- Discussion --- p.38 / Chapter CHAPTER 3 --- ISOLATION OF LECTIN FROM RHIZOMES OF SMILAX GLABRA (FAMILY LILIACEAE) --- p.43 / Chapter 3.1 --- Introduction --- p.43 / Chapter 3.1.1 --- Introduction about Smilax glabra and its chemical constituents --- p.43 / Chapter 3.1.2 --- Introduction about monocot lectins including Liliaceae lectins --- p.45 / Chapter 3.2 --- Materials and methods --- p.50 / Chapter 3.2.1 --- Isolation of lectins from Smilax glabra rhizomes --- p.50 / Chapter 3.2.2 --- Assay for hemagglutinating activity --- p.55 / Chapter 3.2.3 --- Test of inhibition of lectin-induced hemagglutination by various carbohydrates --- p.55 / Chapter 3.2.4 --- "Effects of acid, alkali, temperature and cations on hemagglutinationg activity of lectin" --- p.56 / Chapter 3.2.5 --- Determination of protein concentration --- p.56 / Chapter 3.2.6 --- Molecular mass determination by SDS-PAGE --- p.56 / Chapter 3.2.7 --- Molecular mass determination by gel filtration --- p.56 / Chapter 3.2.8 --- Amino acid sequence analysis --- p.57 / Chapter 3.3 --- Results --- p.57 / Chapter CHAPTER 4 --- ISOLATION OF LECTIN FROM SEEDS OF THE CHINESE CHESTNUT CASTANEA MOLLISIMA (FAMILY FAGACEAE) --- p.74 / Chapter 4.1 --- Introduction to Castanea mollisima and its chemical constituents --- p.74 / Chapter 4.2 --- Materials and Methods --- p.78 / Chapter 4.2.1 --- Isolation of lectin from Chinese chestnuts --- p.78 / Chapter 4.2.2 --- Assay for hemagglutinating activity --- p.83 / Chapter 4.2.3 --- Test of inhibition of lectin-induced hemagglutination by various carbohydrates --- p.83 / Chapter 4.2.4 --- "Effects of acid, alkali, temperature and cations on hemagglutinationg activity of lectin" --- p.83 / Chapter 4.2.5 --- Determination of protein concentration --- p.83 / Chapter 4.2.6 --- Molecular mass determination by SDS-PAGE --- p.83 / Chapter 4.2.7 --- Molecular mass determination by gel filtration --- p.83 / Chapter 4.2.8 --- Amino acid sequence analysis --- p.83 / Chapter 4.3 --- Results --- p.84 / Chapter 4.4 --- Discussion --- p.96 / Chapter CHAPTER 5 --- GENERAL DISCUSSION AND CONCLUSION --- p.98 / REFERENCES: --- p.101

Thesis (M.Sc. (Biochemistry)) -- University of Limpopo, 2012 / Various cytomorphologic and biochemical markers of apoptosis are found in different compartments (plasma membrane, cytoplasm, nucleus, and mitochondria) of target cells. Although the plasma membrane is an easily accessible cellular compartment, relatively little is known about the changes in the expression of plasma membrane glycoproteins during apoptosis, and whether these changes could be used for detection of apoptosis. A critical element of this study was to purify lectins from crude homogenate on glycoprotein-cryogel affinity matrices, and later use the lectins to detect changes on the cell surface of apoptotic cells. Pterocarpus angolensis seed lectin was extracted and fractionated using ammonium sulphate precipitation. The 60 % ammonium sulphate pellet was dissolved in saline azide and purified using Sephadex G-75 affinity chromatography. A 28 kDa lectin was retarded within the column and appeared as a short and broad peak on the chromatogram. Traditionally, Sephadex G-75 column are used predominantly for size exclusion, in this study, the column was used in a non-traditional way for affinity chromatography, as the purified protein is able to bind sugar moieties existing in the structure of Sephadex G-75. A single-step purification of P. angolensis seed lectin was achieved by directly applying unclarified P. angolensis crude extract to the pAAm-cryogel using fetuin as the affinity ligand. Pterocarpus angolensis extract fractionated into 2 peaks, which revealed a highly concentrated band on SDS-PAGE. The results also revealed that an increased binding of the lectin to the fetuin-cryogel matrices was also dependent on the time of incubation. This study suggested very low capacities of the cryogels for the protein due to low coupling sites on the matrix. Taking into account that lectins serve as invaluable tools in diverse area of biomedical research, this study proposed using specific plant lectins to follow the expression of plasma membrane glycoproteins during programmed cell death. Treatment of HL-60 cells with lithium and actinomycin D confirmed a time- and dose-dependent inhibition of proliferation and a decrease in proliferation, which suggest cell death of the treated cells. The observed cell death was further investigated for cellular and biochemical hallmark features of apoptosis, which has shown preferential binding of annexin V-FITC to phosphatidylserine and low molecular DNA ladder. Several FITC labelled lectins were used to detect changes in cell surface glycosylation that accompany apoptosis. This study xvii has shown amongst several FITC-labelled lectins that T. vulgaris lectin could intensively stain the membrane area of apoptotic cells suggesting that the expression of N-acetylglucosamine was significantly increased during actinomycin D induced apoptosis of HL-60 cells. Binding was shown to be specific because it was blocked by the corresponding inhibitory sugar. Thus, the method described in this study could be suitable for the detection of very early stages of apoptosis by recognizing the cell surface carbohydrates of apoptosis.

published_or_final_version / Paediatrics / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Surgery / Doctoral / Doctor of Philosophy

No description available.

No description available.

Recognition of sugar moieties on the surface of microorganisms is one of the ways the body distinguishes potential pathogens from self-cells. The sugarbinding proteins, lectins, mediate this recognition role of the first line of defence against infections, preceding the antibody-mediated (adaptive) immune response. Collectins are calcium-dependent carbohydrate-binding proteins that have been implicated in innate immunity. Bovine conglutinin (BC) and lung surfactant protein-D (SP-D), belong to the family of 'collectins' which are characterised by four domains: an N-terminal cysteine-rich region, a collagenlike region linked with the carbohydrate recognition domain (CRD) via an ahelical neck region. BC and SP-D show remarkable similarity in their amino acid sequence (79% identity), function and biological characteristics. They have been shown to mediate microbial clearance either by directly binding to bacteria leading to phagocytosis or interacting with complement system components. The present study aims to elucidate the biological function of these proteins more precisely. Recombinant fragments (r) of BC and SP-D consisting of their CRDs and neck regions have been cloned in pET-21a and pMal-c2 vectors respectively, for expression in Escherichia coli. Recombinant conglutinin was expressed in BL21(DE3)pLysS and isolated by a denaturation-renaturing procedure. Binding of rBC(N/CRD) to mannan and complement component, iC3b, was assessed in real-time by BIAcore. The dissociation constants were calculated by Scatchard analysis. The carbohydrate structures present on the surface of the microorganisms play an important role in mediating the interactions with the immune cells. The recombinant molecules showed calcium-dependent binding to lipopolysaccharides (LPS) from gram-negative bacteria Pseudomonas aeruginosa, Klebsiella pnuemonia and Salmonella typhosa, which was inhibited in presence of sugars. rBC(N/CRD) also bound to whole bacteria as assessed by ELISA and retained its capacity to recognise various complement system components and the carbohydrate moieties on the surface of various pathogenic microorganisms. The recombinant protein retained its ability to bind various sugar residues, although with lower affinity than that of the native molecule. rBC(N/CRD) is able to bind and aggregate bacteria and cause agglutination of bacterial cell suspensions. A novel model has been used to describe the interactions of the collectins at the molecular level based on specificity of carbohydrate-recognition by the collectins. The pyocin mutant strain 1291 series of Neisseria gonorrhoeae has sequential deletions of the terminal sugars in their lipooligosaccharides (LOS). Conglutinin showed a preferential high affinity binding to 1291a mutant that expresses GlcNAc as the terminal hexose, in comparison to other mutants. This provides a unique system to understand the specific cell-surface interactions in relevance to a particular lectin. Further elucidation of the function of CRD and neck region at a structural level is in progress, using X-ray crystallography. Since the submission of the thesis, the structure of the monomeric CRD has been solved, which revealed a remarkable similarity to the SP-D and MBL structure. Trials are underway to get the structure of the trimeric CRDs. These studies aim to provide a better understanding of the collectinpathogen interaction at the biological and structural levels. The ultimate aim is to determine if the recombinant forms of these proteins can be used therapeutically to enhance the uptake and killing of pathogens.